Insufficient adenosine-induced hyperemia is a major determinant of discordance between non-hyperemic pressure ratio and fractional flow reserve

Adenosine occasionally overestimates fractional flow reserve (FFR) values (i.e., insufficient adenosine-induced hyperemia), leading to low non-hyperemic pressure ratios (NHPR)–high FFR discordance. We investigated the impact of insufficient adenosine-induced hyperemia on NHPR–FFR discordance and the reclassification of functional significance. We measured resting distal-to-aortic pressure ratio (Pd/Pa) and FFR by using adenosine (FFRADN) and papaverine (FFRPAP) in 326 patients (326 vessels). FFRADN overestimation was calculated as FFRADN − FFRPAP. We explored determinants of low Pd/Pa − high FFRADN discordance (Pd/Pa ≤ 0.92 and FFRADN > 0.80) versus high Pd/Pa − low FFRADN discordance (Pd/Pa > 0.92 and FFRADN ≤ 0.80). Reclassification of functional significance was defined as FFRADN > 0.80 and FFRPAP ≤ 0.80. Multivariable analysis identified FFRADN overestimation (p = 0.002) and heart rate at baseline (p = 0.048) as independent determinants of the low Pd/Pa–high FFRADN discordance. In the low Pd/Pa–high FFRADN group (n = 26), papaverine produced a further decline in the FFR value in 21 vessels (81%) compared with FFRADN, and the reclassification was observed in 17 vessels (65%). Insufficient adenosine-induced hyperemia is a major determinant of the low resting Pd/Pa–high FFR discordance. Physicians should bear in mind that the presence of low NHPR–high FFR discordance may indicate a false-negative FFR result.

It has been demonstrated that intravenous adenosine, the vasodilator that is most commonly used for hyperemia induction [1][2][3][4]14 , occasionally fails to induce maximal hyperemia compared to other hyperemic stimuli, such as papaverine [15][16][17][18] . If low NHPR-high FFR discordance is associated with insufficient adenosine-induced hyperemia, vessels that have low NHPR-high FFR discordance may show positive FFR results when another stimulus is used, providing a false-negative result based on an adenosine-induced FFR. Conversely, high NHPR-low FFR discordance may indicate sufficient adenosine-induced hyperemia. The impact of insufficient adenosineinduced hyperemia on NHPR-FFR discordance has not been investigated. Earlier studies used only adenosine for hyperemia induction, thereby precluding an assessment of adenosine's role in NHPR-FFR discordance [19][20][21][22] .
Adenosine produces hyperemia through adenosine A 2a receptors in vascular smooth muscles 4 , whereas papaverine induces maximal hyperemia most reliably by causing a direct relaxation of the vascular smooth muscle 23 . In our present investigation, patients' FFR values were measured using adenosine (FFR ADN ) and papaverine (FFR PAP ). FFR PAP was used as a reference standard of functional significance. We sought to determine the impact of insufficient adenosine-induced hyperemia on resting Pd/Pa-FFR ADN discordance and the reclassification of functional significance.

Methods
Study patients. This retrospective study included 365 patients with chronic coronary syndrome who underwent an FFR assessment for standard clinical indications. If a patient required FFR assessments for two or more vessels, only the first vessel was included in this study. All of the patients were asked to abstain from food and beverages for > 3 h before the catheterization. More prolonged caffeine abstinence was left to the physician's discretion. The exclusion criteria consisted of any contraindications for adenosine or papaverine, patients with severe arrhythmia (e.g., frequent ectopic beats or atrial fibrillation), the presence of significant valvular disease, an ostial lesion, a prior coronary artery bypass graft, and the use of a theophylline-containing medication. Patients with insufficient pressure data quality, including a signal drift value of more than ± 0.03 after the pullback of the pressure wire and inadequate waveform tracings, were also excluded.
The coronary physiology assessment was performed as part of the routine diagnostic coronary angiography procedures for clinical purposes. All methods were performed in accordance with the relevant guidelines and regulations. Written informed consent for the invasive physiology assessment was obtained from all of the patients before the procedure. The Institutional Review Board approved this retrospective study (reference #3234/ Showa University School of Medicine; 31 August, 2021) and waived the requirement of patient approval for the use of patient data and medical records for research.
Coronary physiologic measurements. Coronary angiography was performed in a standard manner for each patient. Intracoronary isosorbide dinitrate (2 mg) was administered before the physiological assessments. With the use of a coronary-pressure guidewire (Philips Volcano or Abbott Vascular) and a 5-or 6-F guiding catheter without side holes, the distal coronary pressure (Pd) and the aortic pressure (Pa) were obtained simultaneously. The patient's resting Pd/Pa ratio was recorded after his/her full recovery from the influence of contrast media, isosorbide dinitrate, or saline flush.
Adenosine was administered continuously via a femoral vein or a large forearm vein at 140 μg/kg/min for > 150 s 3,4,14,24 . In cases in which steady-state hyperemia was not achieved during the adenosine infusion, the infusion was continued for a minimum of 180 s. Papaverine was used as the last agent to obtain a reliable pullback curve, as it induces hyperemia with minimal variations in Pd/Pa 25 . After confirming that Pd/Pa values had returned to the baseline level, with an interval of ≥ 5 min after the termination of adenosine infusion, intracoronary papaverine (8-10 mg in the right coronary artery or 12-15 mg in the left coronary artery) was given through the coronary catheter, followed by 5 mL of saline 14,26 . Approximately 20 s after the papaverine injection, an FFR pullback recording was performed manually, and the presence of pressure-wire drift was checked. Data analysis. Resting Pd/Pa and FFR. Experienced observers blinded to the patients' coronary angiography results and clinical data manually reviewed the pressure recordings. Pressure waveforms from ectopic beats and the adjacent beats were not included in the analysis. Resting Pd/Pa ratio was calculated as the mean Pd to the mean Pa, and ≤ 0.92 was regarded as a positive ratio 5,11 . FFR ADN was measured during the steady-state hyperemic plateau phase > 60 s after the initiation of the adenosine infusion and > 15 s after the transition to hyperemia 18,27 . The lowest Pd/Pa values on a beat-to-beat basis for adenosine and papaverine were regarded as FFR ADN and FFR PAP , respectively [15][16][17][18]28 , and ≤ 0.80 was used as the cut-off for FFR ADN and FFR PAP [1][2][3]29 . The difference in FFR values between adenosine and papaverine was calculated as FFR ADN − FFR PAP 18 . The reclassification of functional significance was defined as FFR ADN > 0.80 and FFR PAP ≤ 0.80 (false-negative by adenosine), and reverse reclassification was defined as FFR ADN ≤ 0.80 and FFR PAP > 0.80 (false-positive by adenosine). Based on the FFR pull-back curve, the physiological pattern of disease was classified as focal, diffuse, or a combination of both (mixed) by the consensus of experienced observers 30 .
We classified the enrolled vessels into four groups according to their resting Pd/Pa and FFR ADN values: (i) high resting Pd/Pa-high FFR ADN (resting Pd/Pa > 0.92 and FFR ADN > 0.80), (ii) high resting Pd/Pa-low FFR ADN (resting Pd/Pa > 0.92 and FFR ADN ≤ 0.80), (iii) low resting Pd/Pa-high FFR ADN (resting Pd/Pa ≤ 0.92 and FFR ADN > 0.80), and (iv) low resting Pd/Pa-low FFR ADN (resting Pd/Pa ≤ 0.92 and FFR ADN ≤ 0.80). We evaluated the clinical and pathophysiological characteristics between the vessels with low resting Pd/Pa-high FFR ADN discordance and the vessels with high Pd/Pa-low FFR ADN discordance, based on a study of NHPR-FFR discordance 22 .
Coronary angiography. Quantitative coronary angiography was performed in optimal projections with a commercially available system (CAAS Workstation version 7.5, Pie Medical Imaging) by independent investigators  Comparison of the two discordant groups. Table 2 summarizes the patient and lesion characteristics in the low Pd/Pa-high FFR ADN and high Pd/Pa-low FFR ADN groups. Diabetes mellitus was significantly more frequent in the low Pd/Pa-high FFR ADN group compared to the high Pd/Pa-low FFR ADN group: 50% (13/26) vs. 26% (11/42), p = 0.046. The low Pd/Pa-high FFR ADN group tended to receive hemodialysis more frequently: 6% (4/26) vs. 2% (1/42), p = 0.067. The LAD location, the quantitative coronary angiography parameters, and the physiological pattern did not differ between the two groups. Heart rate at baseline was significantly higher in the low Pd/Pa-high FFR ADN group compared to the high Pd/Pa-low FFR ADN group: 73 (IQR 61-81) vs. 62 (IQR 57-71), p = 0.008.

Discussion
Our evaluation of resting Pd/Pa and FFR measured using adenosine and papaverine revealed the following: (1) the overestimation of FFR by adenosine (i.e., insufficient adenosine-induced hyperemia) was the strongest determinant of the low Pd/Pa-high FFR ADN discordance, and (2) in two-thirds of the vessels with low Pd/Pa-high FFR ADN discordance, functional significance was reclassified from a negative result by adenosine (FFR ADN > 0.80) to a positive result by papaverine (FFR PAP ≤ 0.80). This study is first to demonstrate that insufficient adenosineinduced hyperemia is a major determinant of NHPR-FFR discordance and to clarify its influence on the reclassification of functional significance. www.nature.com/scientificreports/ Determinants of low NHPR-high FFR discordance. Due to the differences in physiologic backgrounds between resting and hyperemic conditions, the discordance between NHPR and FFR is not surprising. Coronary flow characteristics and/or microvascular resistance were demonstrated to be associated with NHPR-FFR discordance 20,21 . In vessels with preserved microvascular function (i.e., high coronary flow reserve and low microcirculatory resistance), increased coronary flow during hyperemia produces a greater pressure gradient across stenosis compared to vessels with microvascular dysfunction, leading to high NHPR-low FFR discordance. Conversely, in the presence of impaired microvascular function (i.e., low coronary flow reserve and high microcirculatory resistance), the trans-stenotic pressure gradient during hyperemia is less evident than in vessels with preserved microvascular function, leading to low NHPR-high FFR discordance. Interestingly, we observed large overestimations of FFR by adenosine (≥ 0.05, which exceeds 2 standard deviations between repeated FFR ADN measurements) 27 in as many as 42% of the vessels with low Pd/Pa-high FFR ADN discordance, but in only 7% of vessels with high Pd/Pa-low FFR ADN discordance. This result suggests that the standard 140 μg/kg/min dose of intravenous adenosine may not be sufficient to induce maximal hyperemia in the presence of microvascular dysfunction. The microvascular dysfunction in vessels with low NHPR-high FFR discordance described in previous studies might be attributable in part to submaximal adenosine-induced hyperemia. Further research is necessary to address this possibility.
Insufficient adenosine-induced hyperemia due to caffeine remaining in the blood could also account for the low Pd/Pa-high FFR ADN discordance. Caffeine competitively antagonizes adenosine by blocking adenosine A 2a receptor activity 31 . In the presence of serum caffeine, adenosine overestimated FFR in a linear concentration-response manner, compared with papaverine without involving the adenosine receptors 18 . Despite the lack of systematic pre-procedure caffeine abstinence in our present study population, the patient series reflected real-world clinical situations. Matsumoto et al. reported the associations of the duration of caffeine abstinence with serum caffeine level and FFR ADN − FFR PAP 17 . Even after caffeine abstinence for 12-24 h, as recommended by non-invasive imaging guidelines 32,33 , serum caffeine was still detectable in most patients 17 . The mean difference between FFR ADN and FFR PAP (0.02) observed in the present study is similar to that after caffeine abstinence Table 2. Comparison between two discordant groups. Values are expressed as medians (interquartile ranges) or numbers (percentages). eGFR estimated glomerular rate, FFR ADN fractional flow reserve value associated with adenosine, FFR PAP fractional flow reserve value associated with papaverine, LAD left anterior descending coronary artery, N/A not applicable, Pa mean aortic pressure, Pd mean distal coronary pressure.  17 ; however, such strict caffeine control for all patients undergoing invasive angiography is impractical in routine care. Consequently, the frequency of low Pd/Pa-high FFR ADN discordance in the present investigation was consistent with that of the low NHPR-high FFR ADN discordance in earlier investigations that used adenosine or adenosine triphosphate 11,[19][20][21][22] .
Although it is unclear whether the patients abstained from caffeine in the prior studies 11,[19][20][21][22] , caffeine antagonism might have contributed, in part, to their low NHPR-high FFR ADN discordance. Our analyses also identified the patient's heart rate at baseline as an independent determinant of low Pd/ Pa-high FFR ADN discordance. This result is reasonable from a physiological point of view. The resting coronary flow increases with a higher heart rate, producing a larger resting pressure gradient 34 .

Reclassification of functional significance.
Although there is no doubt regarding the revascularization of lesions with both a low NHPR and a low FFR, it remains unclear whether or not lesions with NHPR-FFR discordance should be revascularized. Lee et al. reported that major adverse cardiovascular events were increased only when both NHPR and FFR were positive 35 . Notably, in two-thirds of the present cases of low Pd/Pa-high FFR ADN discordance, the physiological significance was reclassified from a negative result by adenosine to a positive result by papaverine; that is, false-negative FFR results were provided by adenosine. In addition, two-thirds of these false-negative adenosine-induced FFR results were attributed to a large overestimation of FFR by adenosine, i.e., ≥ 0.05. Patients with false-negative results that are due specifically to large overestimations of FFR miss the opportunity to receive benefits from revascularization, which may lead to adverse outcomes. Other investigations have indicated that when the patients are treated with medical therapy alone, their FFR values, even around the cut-off value, demonstrated a continuous relationship with subsequent adverse coronary events 36,37 .
Based on landmark FFR studies (DEFER, FAME I, and FAME II) in which mainly intravenous adenosine was used for hyperemia induction 1,2,38 , the rate of major adverse cardiac events in deferred lesions was considered to be approximately 1% per year 3 . In a recent large-scale prospective observational trial (the J-CONFIRM registry), major adverse cardiac events occurred less frequently, in as few as 0.4% of deferred lesions 39 . Although none of the reports of these trials provided information on serum caffeine levels or the length of caffeine abstinence, the lower incidence of major adverse cardiac events in the J-CONFIRM trial might have occurred in part because hyperemic stimuli other than adenosine (e.g., papaverine or nicorandil) that do not involve the adenosine receptors were used in more than half of their study patients 39 . Further investigation is necessary to confirm the prognostic values of papaverine-and nicorandil-induced FFR.
Insufficient adenosine-induced hyperemia and/or reclassification of functional significance will not be identified unless another hyperemic stimulus is used. Given the present high incidence (two-thirds) of false-negative FFR ADN results, low NHPR-high FFR ADN discordance mismatch may alert operators to insufficient adenosineinduced hyperemia. In the presence of low NHPR-high FFR ADN discordance, the use of other hyperemic stimuli that do not involve the adenosine A 2a receptors (e.g., papaverine and nicorandil) should be considered to avoid misinterpretations of physiological significance.  www.nature.com/scientificreports/ Instead of wire-derived physiological indices, wire-free angiography-derived computational indices of FFR, such as quantitative flow ratio, have been introduced 40 . Quantitative flow ratio was also reported to show discordance with FFR 41 . Considering that both NHPRs and quantitative flow ratio are measured under non-hyperemic conditions, insufficient hyperemia would cause low quantitative flow ratio-high FFR discordance. In other words, all non-hyperemic physiological indices may provide a clue about insufficient hyperemia.
Study limitations. Several limitations should be acknowledged. First, the number of cases of low Pd/Pahigh FFR ADN discordance was relatively small. Second, the prognostic relevance of low Pd/Pa-high FFR ADN discordance and/or reclassification could not be identified in this study, because some of the vessels with low Pd/Pa-high FFR ADN discordance were revascularized based on positive FFR PAP (≤ 0.80) results. Further research is warranted to address whether NHPR-FFR discordance due to insufficient adenosine-induced hyperemia is associated with adverse outcomes. Third, microvascular function was not assessed. Microcirculatory resistance cannot be accurately evaluated by adenosine in the presence of insufficient adenosine-induced hyperemia. Lastly, the order of hyperemic agents was fixed (papaverine last) because papaverine was used to obtain a reliable pullback curve. Although papaverine was administered after confirming that Pd/Pa values had returned to the baseline level, adenosine's carry-over effect cannot be excluded.

Conclusions
Insufficient adenosine-induced hyperemia is a major determinant of the low resting Pd/Pa-high FFR discordance. Physicians should bear in mind that the presence of a low non-hyperemic pressure ratio but a high adenosineinduced FFR may indicate a false-negative FFR result. Table 3. Association with low resting Pd/Pa-high FFR ADN discordance. Values are expressed as medians (interquartile ranges) or numbers (percentages). eGFR estimated glomerular rate, FFR ADN fractional flow reserve value associated with adenosine, FFR PAP fractional flow reserve value associated with papaverine, LAD left anterior descending coronary artery, Pd/Pa distal-to-aortic pressure ratio.

Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.